Nutritionally enhanced plants

ABSTRACT

The present invention relates to the field of improving nutritional content and more particularly the isoflavone content in plants. The invention provides a process for increasing the content of the isoflavone daidzein in selected plants, novel plants produced by this process and products derivable therefrom.

The present application is a U.S. National Phase Application ofInternational Application PCT/EP03/01465 (filed Feb. 13, 2003) whichclaims the benefit of European Patent Application EP 2251404.6 (filedFeb. 28, 2002) all of which are herein incorporated by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates to the field of improving nutritionalcontent in plants. More particularly the invention relates to themodification of selected plants to improve their content of oestrogeniccompounds and to the plants and plant derived products obtainabletherefrom.

BACKGROUND TO THE INVENTION

Isoflavones are a group of oestrogenic compounds which belong to theflavonoid class of plant secondary metabolites. These compounds areproduced naturally in certain plants expressing the enzyme isoflavonesynthase and in particular in leguminous plants. The presence ofisoflavones is known to provide several advantages including thefacilitation of antimicrobial plant defences and establishing bacterialor fungal symbioses within plants as well aiding nitrogen fixation inroot nodules.

In addition to the advantages that are conferred to plants, the dietarypresence of isoflavones is also believed to provide benefits to humanhealth. For example, dietary isoflavones are believed to be effective atreducing the risk of cancer and cardiovascular disease.

At present, in the human diet the only sources of isoflavones arecertain legumes, such as soybean or chickpea. Soy constitutes by far themajor dietary source, however supplementation of food products with soyor soy-extracts may adversely affect the flavour profile. It wouldtherefore be desirable to extend the range of plants or plant tissuescapable of providing an effective source of isoflavones to the humandiet and in particular a source which does not adversely affect theflavour profile of a product.

WO 00/53771 teaches that to form the isoflavone daidzein in transgenicplants that do not possess an isoflavonoid pathway and thus do notproduce isoflavones in nature, it would be necessary to introducetherein three new genes, namely chalcone reductase (CHR) to co-act withchalcone synthase (CHS) to form 2′,4′,4′-trihydroxychalcone, a suitablechalcone isomerase (CHI) to convert 2′,4′,4′-trihydroxychalcone toliquiritigenin, and isoflavone synthase (IFS).

The applicants have now found that the approach disclosed in WO 00/53771does not allow the formation of daidzein in respect of many plants.Furthermore the applicants believe that the transformation of the tomatoplant as exemplified in example 3 of WO 00/53771 is most unlikely toproduce tomatoes with increased levels of daidzein as purported to beachieved therein.

Studies carried out by Yu et al., (Production of isoflavones genisteinand daidzein in non-legume dicot and monocot tissues. Plant Physiol.2000 124:781-793) applied transcription factors C1 and R with theco-introduction of CHR and IFS into non-differentiated Black MexicanSweet (BMS) maize cultures. This approach yielded only trace elements ofdaidzein in a single cell line.

The use of this single cell system in drawing any conclusions relatingto enzymology and regulation of secondary metabolic pathways indifferentiated tissues is recognised in the art as unreliable (StaffordH. A. (1990) CRC Press, Boca Raton, Fla. p. 225-239).

BMS maize cell cultures are undifferentiated and are not active inflavonol biosynthesis.

The objective technical problem to be solved by the present inventiontherefore relates to the need to provide novel plants which comprisesignificantly increased levels of daidzein and/or daidzein derivatives.

It has now been found that the solution to this problem lies in aprocess which selects a non-isoflavone producing plant or part thereofcomprising both active anthocyanin and flavonol pathways and alters saidplant to increase the enzyme activity of chalcone reductase andisoflavone synthase therein.

At the time of filing it was not known that the selection of anon-isoflavone producing plant comprising both active anthocyanin andflavonol biosynthetic pathways in combination with an increase in thesespecific enzyme activities could be used to provide plants withincreased levels of daidzein and/or derivatives thereof.

Definition of Terms

A non-isoflavone producing plant is suitably defined by the absense ofisoflavone synthase enzyme activity which renders the tissues of theplant unable to produce isoflavones. The absence of isoflavone synthaseactivity can be determined by achieving a negative result in a standardenzyme assay as disclosed in Jung et al., (Nature Biotech. Vol. 18 Feb.2000 p. 208-212) incorporated herein by reference.

The term ‘plant or part thereof’ is used herein to refer to an entireplant or differentiated group of cells forming a part thereof. A part ofa plant for the purpose of the invention may relate to leaves, stems,fruit, seeds, flowers, roots, tubers.

The expression ‘increasing’ is used in comparison to an equivalentunmodified plant or part thereof and may be on an absolute dry weightbasis or in relative terms. Except for the modifications introduced bythe process of the invention, this equivalent plant is geneticallyidentical thereto.

Daidzein as used herein is taken to comprise 7,4′-dihydroxyisoflavone.Derivatives of daidzein are taken to comprise those molecules whichresult from the cellular biochemical modification of daidzein.Preferably a daidzein derivative is selected from the group comprisingpterocarpans e.g. medicarpin, glyceollin, isoflavanones e.g. vestitone,rotenoids e.g. munduserone, isoflavans e.g. vestitol,α-methyldeoxybenzoins e.g. angolensin, 2-arylbenzofurans e.g.centrolobofuran, isoflavonols e.g. 7,2′-dihydroxy-4′-methoxyisoflavonol,isoflav-3-enes e.g. haginin, 3-arylcoumarins e.g. glysyrin, coumestanse.g. coumestrol, coumaronochromones e.g. lupinalbin, coumaranochromenee.g. pachyrrhisomene.

Derivatives of daidzein may also result from one or more chemicalprocesses selected from the group comprising methylation, glycosylation,prenylation and ether linkage.

A plant or part thereof that is active in anthocyanin biosynthesis hasan active anthocyanin pathway and is taken to comprise a tissue whichcomprises mRNA encoding one or more enzymes selected from the groupcomprising dihydroflavonol reductase, proanthocyanidin synthase, andUDP-glucose:flavonoid-3-O-glucosyltransferase.

For the purpose of the present invention active anthocyanin biosynthesismay be determined in a plant tissue by a spectrophotometric test whereinabsorbance of a hydrolysed plant extract at λvis-max 480-560 nm isindicative of an active anthocyanin pathway. Plant tissues are ground inliquid nitrogen and extracted with 80% (v/v) ethanol at 100 mg/700 μlfor 30 min at room temperature (˜22/C). Following extraction the celldebris is removed by filtration through a 0.45 μm Millex_HV filter unit(Millipore Corp, USA). The ethanol extract (360 μl) is mixed with 12MHCl (40 μl). The acidified ethanol extract is assayed byspectrophotometer and absorbance determined as A_(λvis-max 480-560nm),with the A₆₅₇ subtracted.

It is preferred that a plant or the part thereof that is active inanthocyanin biosynthesis contains more than 10 mg/kg fresh weightanthocyanin, more preferably at least 100 mg/kg, further preferred atleast 1000 mg/kg and most preferred from 1000 to 10,000 mg/kg freshweight. Suitably this is calculated from absorption values according theformula C=A*MW*10³*DF/(ε*1) in which C refers to concentration, A refersto absorption (as defined above); MW is molecular weight; DF is dilutionfactor; ε is molar extinction coefficient (29,600 for cyanidin3-glucoside the major anthocyanin in nature) and l is the path length.

A plant or part thereof which is active in flavonol biosynthesis has anactive flavonol pathway and is taken to comprise any tissue whichcomprises mRNA encoding one or more enzymes selected from the groupcomprising chalcone synthase, chalcone isomerase, flavanone3-hydroxylase, flavonol synthase.

For the purpose of the present invention whether a plant is active inflavonol biosynthesis may be determined by preparing a hydrolysed tissueextract and detection by HPLC analysis.

For extraction, tissues are harvested and flash frozen in liquidnitrogen before being stored at −80° C. The tissues are then ground to afine powder to ensure a homogeneous mix. An aliquot from this mixture isthen extracted for 30 min at room temperature (˜22° C.) in 80% (v/v)ethanol at 100 mg/700 μl. Following extraction, the cell debris isremoved by filtration through a 0.45 μm Millex-HV filter unit (MilliporeCorp, USA). The filtrate is stored at −20° C. prior to HPLC analyses.

For hydrolysed extracts, 40 μl of 12M HCl is added to 360 μl from eachtissue extract, before incubating at 90° C. for 40 min.

After hydrolysis, an aliquot from each extract is filtered through a 0.2μm PTFE disposable filter (Whatman). The filtrate (20 μl) is injectedinto the HPLC system (HP1100, Agilent) via an autosampler maintained at4° C. The analytical column (Prodigy Phenyl-3, 4.6×150 mm, particle size5 μm, (Phenomenex) is held at 30° C. Detection is by diode array,monitoring at 262, 280, and 370 nm. Observed peaks are scanned from210-550 nm to obtain spectra. Chemstation software (Rev. A.8.03) wasused to control the system and collect and analyse data.

Absorbance spectra (corrected for baseline spectrum) and retention timeof peaks are compared with those of commercially available flavonolstandards to determine whether the plant tissue is active in flavonolbiosynthesis.

It is preferred that a plant or the part thereof that is active inflavonol biosynthesis contains at least 10 mg/kg fresh weight offlavonol, preferably at least 100 mg/kg more preferred at least 1000mg/kg, most preferred from 1000 to 10000 mg/kg.

A ‘functional equivalent’ nucleotide sequence is any sequence whichencodes a protein which performs the same biological function.

According to another embodiment, a functionally equivalent nucleotidesequence shows at least 50% identity to the respective DNA sequence.More preferably a functionally equivalent DNA sequence shows at least60%, more preferred at least 75%, even more preferred at least 80%, evenmore preferred at least 90%, most preferred 95-100% identity, to therespective DNA sequence (DNAStar MegAlign Software Version 4.05 and theClustal algorithm set to default parameters).

According to a further preferred embodiment a functionally equivalentsequence shows not more than 5 base pairs difference to the respectiveDNA sequence, more preferred less than 3, e.g. only 1 or 2 base pairsdifferent.

According to another embodiment a functionally equivalent sequence iscapable of hybridising under low stringent (2×SSC, 0.1% SDS at 25° C.for 20 min) conditions to the respective sequence, more preferably afunctionally equivalent sequence is capable of hybridising under mediumstringent conditions (1×SSC, 0.1% SDS, 25° C. for 20 min), furtherpreferred a functionally equivalent sequence is capable of hybridisingunder high stringent conditions (0.1×SSC, 0.1% SDS, 25° C. for 20 min).

Preferably an equivalent DNA sequence is capable of transcription andsubsequent translation to an amino acid sequence showing at least 50%identity to the amino acid sequence encoded by the respective DNAsequence. More preferred, the amino acid sequence translated from anequivalent DNA sequence has at least 60%, more preferred at least 75%,even more preferred at least 80%, even more preferred at least 90%, mostpreferred 95-100% identity to the amino acid sequence encoded by therespective DNA sequence (DNAStar MegAlign Software Version 4.05 and theClustal algorithm set to default parameters.)

BRIEF DESCRIPTION OF THE INVENTION

It has now been found that novel plants which comprise significantlyincreased levels of daidzein and/or daidzein derivatives may be providedby a plant or part thereof which has both active anthocyanin andflavonol pathways and has been genetically modified to increase theenzyme activity of CHR and IFS therein.

It is a therefore a first object of the invention to provide agenetically modified plant or part thereof comprising daidzein and/orderivatives thereof, wherein said plant or part thereof is active inflavonol and anthocyanin biosynthesis and comprises one or morenucleotide sequences encoding chalcone reductase and one or morenucleotide sequences encoding isoflavone synthase.

Particular advantage has been found to result from genetically modifyingsaid plant to also increase the activity CHI, wherein production ofdaidzein in the tissues concerned showed a 90 fold increase over themodification to increase CHR and IFS activities alone. This additionalbenefit has been found to be dependent on the selection of a CHI isoformwhich is capable of catalysing the conversion of4,2′,4′-trihydroxychalcone to 7,4′-dihydroxyflavanone (liquiritigenin).Suitable CHI's are obtained from leguminous plants. At some 15 times (ona dry weight basis) the level of daidzein present in conventional soythis result represents a significant improvement to the art. This resultis surprising and clearly demonstrates a synergy from a combinedincrease in these three enzyme activities where active anthocyanin andflavonol pathways are present in a plant.

A first embodiment of the invention therefore comprises a geneticallymodified plant or part thereof as described above, further comprisingone or more nucleotide sequences encoding a chalcone isomerase capableof catalysing the conversion of 4,2′,4′-trihydroxychalcone to7,4′-dihydroxyflavanone.

A further embodiment of the invention comprises a genetically modifiedplant or part thereof as described above wherein said one or morenucleotides sequences comprise sequences according to sequenceidentification numbers 1 and 3, or functional equivalents thereof.

In the preferred instance where the synergistic advantage of anadditional CHI increase is sought, the invention relates to anembodiment wherein said one or more nucleotides sequences comprisessequences according to sequence identification numbers 1, 3 and 5, or afunctional equivalents thereof.

In a further embodiment the invention relates to a genetically modifiedplant or part thereof as described above wherein said plant or partthereof is selected from the group comprising, but not restricted to,tobacco, Lactuca sp., broccoli, asparagus, red cabbage, potato, spinach,rhubarb, red onion, shallot, aubergine, radish, Swiss chard, purplebasil, watermelon and berries.

Plants or parts thereof modified in accordance with the invention toincrease their content of daidzein and/or daidzein derivatives areparticularly advantageous for providing health benefits associated withincreased dietary uptake of isoflavones. Plants or parts thereof maytherefore be used either in their natural state or prepared as a extractto treat disease states or induce health benefits as a preventativeagent or by counter acting the ageing process.

A second object of the invention therefore provides an extract of aplant as described above wherein said extract comprises daidzein orderivatives thereof.

A third object of the invention provides an extract as described abovefor use as a medicament. In a preferred embodiment and extract accordingto the invention may be used in the treatment and/or prevention of oneor more conditions selected from the group comprising, osteoporosis;cancer; menopausal and post menopausal symptoms comprising hot flushes,anxiety, depression, mood swings, night sweats, headaches, unrinaryincontinence; pre-menstrual syndromes comprising fluid retention,cylical mastalgia, dysmenorrhoea; heart disease atherosclerosis;hypertension; coronary artery spasm; high cholesterol; Alzheimer'sdisease; impaired cognitive function; inflammatory diseases comprisinginflammatory bowel disease, ulcerative colitis, Crohn's disease; andrheumatoid arthritis.

Cosmetic benefits may also be gained from the use of an extract asdescribed above in the treatment and/or prevention of one or moreconditions selected from the group comprising sunlight induced skindamage, skin wrinkling, loss of skin sensitivity, loss of skin firmness,acne, poor hair condition and baldness.

These medical and cosmetic benefits are also provided by the use of thegenetically modified plants or parts thereof according to the invention.Uptake of the daidzein and/or its derivative may be via oral or topicalapplications.

For convenient dietary uptake of increased amounts of isoflavones aplant or part thereof according to the invention may suitably beincorporated into a food product or nutritional supplement. Hence, afurther object of the invention provides for the use of a geneticallymodified plant or part thereof or of an extract as described in a foodproduct or nutritional supplement.

It is to be noted that unlike conventional soy derived sources ofdietary isoflavones, daidzein and the derivatives thereof as provided byplants according to the invention may be incorporated into food productswithout adversely affecting the flavour profile of such products. Inthis way an additional problem in the art is solved by the presentinvention.

A further object of the invention is therefore to provide a food productcomprising a genetically modified plant or part thereof according to thedescription above. Preferably a food product according to the inventionwill be frozen to allow the content of daidzein and/or its derivativesto remain stable on storage.

In a most preferred embodiment a food product according to the inventionis selected from the group comprising pre-packed mixed salads, soups,spreads, sauces, fruit/cereal bars and ice creams.

A nutritional supplement comprising an extract of a plant or partthereof as described above is also provided by the present invention.

It is a further object of the invention to provide a process forincreasing the content of daidzein and/or derivatives thereof in a plantor part thereof, wherein said process comprises the steps;

-   (i) selecting a non-isoflavone producing plant wherein said plant or    part thereof is active in anthocyanin and flavonol biosynthesis;-   (ii) genetically modifying said plant to increase the activity of    chalcone reductase and isoflavone synthase in said plant or part    thereof.

In a first embodiment, the process of the invention further comprisesgenetically modifying said plant or part thereof to increase theactivity of a chalcone isomerase wherein said chalcone isomerase iscapable of catalysing the conversion of 4,2′,4′-trihydroxychalcone to7,4′-dihydroxyflavanone. In this way the process achieves thesynergistic increase in the content of daidzein and/or its derivatives.

A further embodiment wherein the activity of chalcone reductase andisoflavone synthase are to be increased comprises a process as disclosedabove wherein one or more nucleotide sequences according to sequenceidentification numbers 1 and 3, or functional equivalents thereof arestably integrated into the genome of said plant.

To achieve the desired increase in activity of chalcone isomerase apreferred embodiment comprises a process as disclosed above additionallycomprising stably integrating into the genome of said plant one or morenucleotide sequence according to sequence identification number 5, orfunctional equivalents thereof.

In a most preferred embodiment the process according to the inventionrelates to a plant or part thereof selected from the group comprising,but not resticted to, tobacco, Lactuca sp., broccoli, asparagus, redcabbage, potato, spinach, rhubarb, red onion, shallot, aubergine,radish, Swiss chard, purple basil, watermelon and berries such asstrawberries.

DETAILED DESCRIPTION OF THE INVENTION

A sequence encoding a biosynthetic enzyme for increasing the tissuecontent of daidzein and/or daidzein derivatives may be a genomic or cDNAclone, or a sequence which in proper reading frame encodes an amino acidsequence which is functionally equivalent to the amino acid sequence ofthe biosynthetic gene encoded by the genomic or cDNA clone. A functionalderivative can be characterised by an insertion, deletion or asubstitution of one or more bases of the DNA sequence, prepared by knownmutagenic techniques such as site-directed mutagenesis or derived from adifferent species.

For the performance of the present invention any nucleotide sequencesencoding an enzyme with the biological function of a chalcone reductase,isoflavone synthase or chalcone isomerase may be used in thetransformation of a suitably selected plant to increase these enzymeactivities with said plant or part thereof.

Biological function of a nucleotide sequence encoding a chalconereductase can be assessed by a standard assay (Welle et al., 1988 FEBSletter 236:221-225; Welle et al., 1991 Eur J Biochem 196:423-430; Welleand Schroder, 1992 Arch. Biochem. Biophys 293:377-381). To obtainprotein, the nucleotide sequence is sub-cloned into a prokaryoticexpression vector, such as pTZ19R (Pharmacia), and transformed intoEscherichia coli. Selected E. coli clones harbouring the nucleotidesequences of interest are grown to a culture density of A₆₀₀=0.6-1before inducing expression with 1 mM isopropyl β-D-thiogalctopyranoside(IPTG) for 2.5 hours. Following induction, bacteria are harvested bycentrifugation and resuspended in 0.1M potassium phosphate, 0.6 mg/mllysozyme and 1.2M EDTA and placed on ice for 45 min to lyse. The lysateis centrifuged at 16000 g for 20 min and an aliquot of supernatant usedin the chalcone reductase assay.

Chalcone reductase activity is assayed in a final volume of 120 μl,comprising 80 μl chalcone reductase protein extract, 10 μmol potassiumphosphate pH 5.0, 0.12 μmol NADPH, 1 nmol 4-coumaroyl CoA, 1.5 nmol[2-¹⁴C] malonyl-CoA, 22.2 fkat pure soybean CHS (˜3 μg). Reactions arerun for 60 min at 30° C. before the reaction products are extracted in200 μl ethyl acetate. The organic phase is separated by centrifugation,concentrated in vacuo and separated by thin layer chromatography using15% acetic acid (presence of chalcone isomerase) or CHCl₃/aceticacid/water (10:9:1) (absence of chalcone isomerase). The identity of6′-deoxychalcone is established by co-chromatography with a referencesample.

Suitable CHR encoding sequences already known in the art comprise;Alfalfa (Medicago sativa): accession numbers CHR1a-X82366, CHR1b-X82367,CHR2a-X82368, CHR7-U13925, CHR12-U13924; Chickpea (Cicer arietinum)accession number AB024989; Soybean (Glycine max) accession numberX55730; Liquorice (Glycyrrhiza glabra) accession numbers CHRa-D86558,CHRb-D86559;

Alternatively suitable CHR encoding sequences may be isolated from otherspecies. Sequence alignment of CHR's already known in the art, show twoconserved regions Met-Pro-Val-Val-Gly-Met-Gly-Ser-Ala (Seq. ID No.7) andAla-Ile-Ile-Glu-Ala-Ile-Lys-Gln (Seq. ID. No. 8) identified toward the5′ end of the coding sequence. Degenerate primers 327 and 328 (see FIG.4) are designed to each of these coding sequences respectively.Sequences encoding CHR are isolated by polymerase chain reaction usingprimers 327 and 328 in conjunction with a dT₁₇ primer and using a 3′cDNA library target. The resulting fragments were cloned into a pT7vector and sequenced. Alignment of these sequences with those known inthe art would allow provisional identification. To obtain full-lengthcoding sequence, 5′ and 3′ sequence can be obtained using standard5′RACE and 3′RACE procedures as disclosed in example 1 (1.3.3).

A nucleotide sequence encoding an enzyme with isoflavone synthaseactivity may also be determined by a standard assay, wherein yeastmicrosomes are prepared from control WHT1 and strains expressing acytochrome P450 cDNA according to the methods of Pompon et al., (MethodsEnzymol. 272, 51-64). The assay is carried out according to Jung et al.,(Nature Biotech 2000, vol 18 February 200, p208-212). The proteincontent of each microsome preparation is assayed using the Bradfordprotein micro assay (Bio-Rad. Hecules. CA). From 30 to 150 μg ofmicrosomal proteins are incubated at room temperature in 80 mM K₂HPO₄,0.5 mM glutathione. 20% (wt/vol) sucrose, pH 8.0 with 100 μM naringeninor 100 μM liquiritigenen substrate and 40 nmol of NADPH added per each100 μl of final reaction volume. Following incubation, reactions areextracted with ethyl acetate. Samples assayed on a Hewlett-Packard 1100series HPLC system using either a LiChrospher RP-C18 column (5 m 250×3mm) or a Phenomenexz Luna C18 (2) column (3 u; 150×4.6 mm).

On the first column samples in ethyl acetate of candidate cDNA assaysare isocratically separated for 5 min employing 65% methanol as a mobilephase. For the second column samples are evaporated and resuspended in80% methanol and then separated using a 10 min linear gradient from 20%methanol/80% 10 mM ammonium acetate, pH 8.3 to 100% methanol at a flowrate of 1 ml min⁻¹ or using 65% methanol as mobile phase for isocraticelution. Genistein and daidzein are monitored by the absorbance of 260nm. Using authentic naringinen, liquiritigen, genistein and daidzein(Indofine Chemical, Somerville NI) dissolved in ethanol as standards forcalibration peak areas are converted to nanograms.

To confirm the identity of genistein and daidzein, samples areevaporated and resuspended in 25% acetonitrile in water and assayed on aHewlett-Packard/Micromass LC/MS by running 25 μl on a Zorbax EclipseXDB-C8 reverse-phase column (3×150 mm 3.5 μm) isocratically with 25%solvent B (0.1% formic acid in acetonitrile) in solvent A (0.1% formicacid in water). Mass spectrometry is done by electrospray scanning from200 to 400 m/e, using −6 volt cone voltage. The diode array signals weremonitored between 200 and 400 nm in both instruments.

Suitable IFS sequences already known in the art include Mung Beanaccession number AF195807; Red Clover accession number AF195811; andSnow Pea accession number AF195812.

Alternatively suitable IFS cDNAs may be isolated from other species.Jung et al. (Nature Biotech 2000, vol 18 February 200, p208-212)describe how mung bean sprouts and snow pea sprouts were obtained fromthe grocery store. Seeds for alfalfa, red clover, white clover, hairyvetch and lentil can be obtained from Pinetree Garden Seeds (NewGloucester, Me.) seeds for lupine cv. Russel Mix were obtained fromBotanical Interests (Boulder, Colo.), and seeds for sugarbeet wereobtained from a commercial source.

Seedlings were grown and RNA prepared using TRIzol Reagent (Gibco BRL)and first-strand cDNA was prepared as described above. OligodT was usedas the reverse transcription primer in all cases except with whiteclover for which random hexamers were used as the reverse transcriptionprimer: Polymerase chain reaction amplifaction was carried out usingAdvantage-GC cDNA polymerase mix (Clontech) using primer set one5′ATGTTGCTGGAACTTGCACTT-3′ (Seq ID. No. 9) and5′TTAGAAAGGAGTTTAGATGCAACG-3′ (Seq. ID. No. 10) or the nested primer settwo: 5′TGTTTCTGCATTGCGTCCCAC-3′ (Seq. ID. No. 11) and5′-CCGATCCTTGCAAGTGGAACAC-3′ (Seq. ID. No. 12) as follows: Mung bean andred clover PCR products amplified using primer set one were cloneddirectly into pCR2.1.

For white clover, lentil, hairy vetch, alfalfa, lupine, and beet a firstPCR with primer set one was followed by a second primer set two, and theresulting fragments cloned. For snow pea, a first PCR with primer setone was followed by a second PCR with high annealing temperature (60°C.) using primer set one. The expected size product was gel purified andused as a template in a third PCR with the high annealing temperatureand primer set one. The resulting product was cloned into pCR2.1. AllPCR fragments in pCR2.1 were sequenced. All alignments were carried outusing DNAStar MegAlign software version 4.05 and the Clustal algorithmset to default parameters.

The coding regions for accession numbers AF195807 (mung bean). AF195811(red clover), and AF195812 (snow pea) were amplified and cloned intopRS315-gal using “gap repair” and microsomes were produced and assayedas described above.

A nucleotide sequence encoding an enzyme with chalcone isomeraseactivity capable of catalysing the conversion of4,2′,4′-trihydroxychalcone to 7,4′-dihydroxyflavanone may be determinedby a standard assay (Dixon et al., 1982 Biochem. Biophys Acta 715:25-33; Mol et al., 1985 Phytochemistry 24: 2267-2269, Terai et al., 1996Protein Expression and Purification 8:183-190). To obtain protein, thenucleotide sequence is sub-cloned into a prokaryotic expression vector,such as pET vectors (Invitrogen), and transformed into Escherichia coli.Selected E. coli clones harbouring the nucleotide sequences of interestare grown to a culture density of A₆₀₀=0.6-1 before inducing expressionwith 1 mm isopropyl β-D-thiogalctopyranoside (IPTG) for 2.5 hours.

Following induction, bacteria are harvested by centrifugation andresuspended in 0.1M potassium phosphate, 0.6 mg/ml lysozyme and 1.2MEDTA and placed on ice for 45 min to lyse. The lysate is centrifuged at16000 g for 20 min and an aliquot of supernatant used in the chalconeisomerase assay.

Chalcone isomerase activity is assayed in a final volume of 1 ml,comprising either 18.4 μm tetrahydroxychalcone (naringenin chalcone) or12.7 μg trihydroxychalcone (isoliquiritigenin) substrate, chalconeisomerase protein extract, 5% (w/v) bovine serum albumin and 0.1Mpotassium phosphate buffer (pH5.8). Chalcone isomerase activity againstboth tri- and tetra-hydroxychalcone substrates is detected by a decreasein absorption at 385 nm.

Suitable CHI sequences already known in the art comprise those derivedfrom; French bean (Phaseolus vulgaris) accession number X16470; Kudzuvine (Pueraria montana var. lobata): accession number D63577; Soybean(Glycine max): accession number AF276302; Alfalfa (Medicago sativa):accession number M910079; Garden Pea (Pisum sativum): accession numberU03433.

Alternatively the well-established correlation between CHI function andstructure enables suitable CHI sequences to be isolated from othersources. Numerous cloning strategies have been shown in the art to beeffective at isolating CHI cDNAs and may be adopted by the personskilled in the art to identify alternative CHI encoding sequences.

Shirley, B. W., et al., (Plant Cell, Vol. 4, 333-347 1992) describes aPCR based approach to obtaining CHI cDNA from Arabidopsis wherein theidentification of consensus sequences for primer design as well as PCRreaction conditions are disclosed. Sparvoli, F. et al., (Plant Mol.Biol. 24: 743-755, 1994) describes the cloning of CHI from a cDNAlibrary by using heterologous Antirrhinum CHI cDNA probes. Grotewold E.et al., (Mol. Gen. Genet. (1994) 242: 1-8) describes the isolation andcharacterisation of a maize gene encoding CHI, the cloning strategy andsuitable primers.

The literature outlined above clearly demonstrates that correspondingCHI sequences from other plants; alternative cloning strategies forother CHI genes; knowledge of consensus sequences for the generation ofprimers; appropriate PCR conditions are known in the art. The personskilled in the art is therefore able to identify and use alternative CHIsequences for the transformation according to the present invention.

Gene constructs according to the invention either comprise one or morenucleotide sequences encoding chalcone reductase and isoflavonesynthase, or comprise one or more nucleotide sequences encoding chalconereductase, isoflavone synthase and chalcone isomerase depending on themagnitude of increase sought.

The gene sequences of interest will be operably linked (that is,positioned to ensure the functioning of) to one or more suitablepromoters which allow the DNA to be transcribed. Suitable promoters,which may be homologous or heterologous to the gene (that is, notnaturally operably linked to a gene encoding an enzyme for flavonoidbiosynthesis), useful for expression in plants are well known in art, asdescribed, for example, in Weising et al., (1988) Ann. Rev. Genetics22:421-477. Promoters for use according to the invention may beinducible, constitutive, or tissue-specific or have various combinationsof such characteristics.

Useful promoters include, but are not limited to constitutive promoterssuch as carnation etched ring virus (CERV) promoter, cauliflower mosaicvirus (CaMV) 35S promoter, or more particularly the enhanced cauliflowermosaic virus promoter, comprising two CaMV 35S promoters in tandem(referred to as a “Double 35S” promoter). These would have the effect ofincreasing isoflavonoid levels throughout a plant.

Accordingly, the invention provides in a further aspect a gene constructin the form of an expression cassette comprising as operably linkedcomponents in the 5′-3′ direction of transcription, one or more unitseach comprising a suitable promoter in a plant cell, a plurality ofnucleotide sequences selected from the group comprising sequencesencoding a CHR and IFS and a suitable transcriptional and translationaltermination regulatory region. More preferably said group comprisessequences encoding CHR, IFS and a CHI capable of catalysing theconversion of 4,2′,4′-trihydroxychalcone to 7,4′-dihydroxyflavanone.

The promoter and termination regulatory regions will be functional inthe host plant cell and may be heterologous or homologous to the plantcell and the gene. Suitable promoters which may be used are describedabove.

The termination regulatory region may be derived from the 3′ region ofthe gene from which the promoter was obtained or from another gene.Suitable termination regions, which may be used, are well known in theart and include Agrobacterium tumefaciens nopaline synthase terminator(Tnos), Agrobacterium tumefaciens mannopine synthase terminator (Tmas),the rubisco small subunit terminator (TrbcS) and the CaMNV 35Sterminator (T35S). Particularly preferred termination regions for useaccording to the invention include the Tnos and TrbcS terminationregions.

Such gene constructs may suitably be screened for activity bytransformation into a host plant via Agrobacterium tumefaciensco-transformation and screening for daidzein levels.

Conveniently, the expression cassette according to the invention may beprepared by cloning the individual promoter/gene/terminator units into asuitable cloning vector. Suitable cloning vectors are well known in theart, including such vectors as pUC (Norrander et al., (1983) Gene26:101-106), pEMBL (Dente et al., (1983) Nucleic Acids Research11:1645-1699), pBLUESCRIPT (available from Stratagene), pGEM (availablefrom Promega) and pBR322 (Bolivar et al., (1977) Gene 2:95-113).Particularly useful cloning vectors are those based on the pUC series.The cloning vector allows the DNA to be amplified or manipulated, forexample by joining sequences. The cloning sites are preferably in theform of a polylinker, that is a sequence containing multiple adjacentrestriction sites, to allow flexibility in cloning.

Preferably the DNA construct according to the invention is comprisedwithin a vector, most suitably an expression vector adapted forexpression in an appropriate host (plant) cell. It will be appreciatedthat any vector which is capable of producing a plant comprising theintroduced DNA sequence will be sufficient.

Suitable vectors are well known to those skilled in the art and aredescribed in general technical references such as Pouwels et al.,Cloning Vectors. A laboratory manual, Elsevier, Amsterdam (1986).Particularly suitable vectors include the Ti plasmid vectors.

Transformation techniques for introducing the DNA constructs accordingto the invention into host cells are well known in the art and includesuch methods as micro-injection, using polyethylene glycol,electroporation, or high velocity ballistic penetration. A preferredmethod for use according to the present invention relies onAgrobacterium tumefaciens mediated co-transformation.

After transformation of the plant cells or plant, those plant cells orplants into which the desired DNA has been incorporated may be selectedby such methods as antibiotic resistance, herbicide resistance,tolerance to amino-acid analogues or using phenotypic markers.

Various assays within the knowledge of the person skilled in the art maybe used to determine whether the plant cell shows an increase in geneexpression, for example, Northern blotting or quantitative reversetranscriptase PCR (RT-PCR). Whole transgenic plants may be regeneratedfrom the transformed cell by conventional methods. Such transgenicplants having improved daidzein levels may be propagated and crossed toproduce homozygous lines. Such plants produce seeds containing the genesfor the introduced trait and can be grown to produce plants that willproduce the selected phenotype.

Plants or parts thereof which have been modified in accordance with thepresent invention may be the used as a source of daidzein and/or one ormore of its derivatives in the form of an enriched extract or asubstantially pure form.

Food products which comprise the plants, plant parts or extracts thereofin accordance with the present invention enable the consumer to takefull advantage of the health benefits associated with increasedisoflavone uptake while at the same time avoiding the adverse flavourassociated with soy derived isoflavones in the prior art.

Salad leaves are particularly suited to genetic transformation by theprocess of the invention and therefore species of lettuce (Lactuca sp.)such as Lactuca sativa e.g. ‘Red Oak Leaf’, ‘Red Leprechaun’; Lactucasativa group Butterhead lettuce e.g. Mira, Redcross; Lactuca sativagroup Cos lettuce e.g. ‘Romaine Red Cos’, Four Seasons Red’, Seville;Lactuca sativa group Crisp lettuce e.g. ‘Red Salad Bowl’, Red Grenoble’;Lactuca sativa group Cutting lettuce e.g. ‘Lollo Rosso’, Revolutiontransformed in accordance with the present invention provide a idealmeans of supplementing dietary needs and may be provided washed andpre-packed to the consumer.

Fruit containing snack bars or breakfast cereals provide a convenientmeans of supplementing the human diet with isoflavones. Fruit piecesderived from a plant according to the invention are suitably dried tofrom 10 to 90%, preferably 20 to 60%, most preferably about 40% of theirfresh weight to give shelf stability and incorporated into a bar orcereal product.

Fruits with high levels of daidzein and/or daidzein derivatives inaccordance with the invention are also be ideally incorporated intoyoghurts and ice creams or to flavour fruit drinks.

Suitable fruits for these food products would include raspberries,strawberries, blackcurrants, red currants, blueberries and blackberries.

Plants or parts thereof which have been genetically modified inaccordance with the present invention may also provide a source of anextract rich in daidzein and/or its derivatives or a purified formthereof for inclusion in products such as nutritional supplements,calorie controlled drinks and low fat spreads.

A large body of evidence supports the cosmetic and medical healthbenefits that can be attributed to human dietary consumption ofisoflavones and in particular daidzein. These include: activity as bothestrogenic and anti-estrogenic agents (Coward et al., 1993; Martin, etal., 1996); anticancer effects associated with phytoestrogenic activity(Lee et al., 1991; Adlercreutz et al., 1991); anticancer effectsassociated with inhibition of several enzymes including DNAtopoisomerase and tyrosine protein kinase (Akiyama, et al., 1987; Uckun,et al., 1995); suppression of alcohol consumption (Keung and Vallee,1993; Keung et al., 1995); antioxidant activity (Arora et al., 1998;Tikkanen et al., 1998); increasing bone remineralisation (Tomonaga etal., 1992; Draper et al., 1997); and beneficial cardiovascular effects(Wagner et al., 1997).

The present invention may be more fully understood by reference to theaccompanying figures in which:

FIG. 1: shows the pea chalcone reductase DNA sequence (SEQ ID No. 1) andits corresponding protein sequence (SEQ ID No. 2).

FIG. 2: shows the soy isoflavone synthase DNA sequence (SEQ ID No.3) andits corresponding protein sequence (SEQ ID No. 4)

FIG. 3: Lotus corniculatus chalcone isomerase DNA sequence (SEQ ID No.5) and its corresponding protein sequence (SEQ ID No. 6)

FIG. 4: provides primer sequences used in accordance with the invention.

FIG. 5: illustrates Plasmid maps of pPV5LN, pPE2, pPE5, pPE9, pPE11,pPE15, pPE51, pPE120 and pPE125.

FIG. 6: illustrates GC-MS analysis of tobacco petal extracts fromrepresentative tobacco transfomants, pPE120/24, pPE120/26 and pPE51spiked with an authentic daidzein standard. A. Peak with retention timecorresponding with authentic daidzein (RT=19.60) is present I pPE120/24and pPE120/26 transformants. B. Selected ion monitoring of pPE120/24 andpPE51/9 spiked with an authenitc daidzein standard shows characteristicpeaks in pPE120/24.

FIG. 7: illustrates accumulation of daidzein in petal tissue fromtobacco transformants harbouring constructs encoding chalcone reductaseand isoflavone synthase (pPE120) activitites with controls (pPE51).Ethanol extracts from petals were hydrolysed and analysed by HPLC.

FIG. 8: illustrates accumulation of daidzein in petal tissue fromtobacco transformants harbouring constructs encoding chalcone reductase,chalcone isomerase and isoflavone synthase (pPE125) activitites withcontrols (pPE51). Ethanol extracts from petals were hydrolysed andanalysed by HPLC.

EXAMPLE 1 cDNA Cloning of Chalcone Reductase, Chalcone Isomerase andIsflavone Synthase and the Generation and Analysis of Transgenic N.tabacum

1.1 Plant Material

All experiments can be performed using normally available tobacco(Nicotiana tabacum) genotypes as the starting material. N. tabacumcultivar SR1 is such a genotype. Plants of N. tabacum cultivar SR1 weregrown in controlled temperature growth rooms with a 16-hour photoperiodat a temperature of 25° C.

1.2 Bacterial Strains

Escherichia coli strain XL1-Blue: recA1, endA1, gyrA96, thi-1, hsdR17,supE44, relA1 lac [F′proAB laCl^(q)ZΔM15 Tn10 (Tet^(r))] (StratageneEurope, The Netherlands). Transformation of E. coli XL1-Blue wasperformed using the method of Hanahan (1983). Agrobacterium tumefaciensLBA4404 (Hoekema, 1985). Transformation of Agrobacterium tumefaciensLBA4404 was performed according to Shen and Forde (1989).

1.3 Gene Cloning

1.3.1 Total RNA Isolation

RNA was isolated from Lotus corniculatus (Lotus), Glycine max (soybean),Pisum sativum (pea) and Medicago sativa (alfalfa) leaf tissue using aPurescript RNA isolation kit (Pharmacia) according to manufacturer'sinstructions.

1.3.2 cDNA Synthesis

5′ cDNA library construction: 2 μg of RNA isolated from either Lotus,soybean, pea or alfalfa tissue was heated to 65° C. for 10 minutes, thensnap cooled on ice. The RNA was reverse transcribed in a 20 μl reactionfor 90 minutes at 42° C. using 10 units of stratascript (Gibco-BRL) in1×rt buffer (Gibco BRL), 30 mM dNTPs (DATP, dCTP, dTTP, dGTP)(Pharmacia), 0.1M DTT, 1 U/μl RNasin (Roche) and 50 pmoles randomhexamers. The random primed cDNA was then purified using a Gibco-BRL pcrpurification kit (according to manufacturer's instructions). Thepurified cDNA was then poly A tailed in 50 μl of 1× tailing buffer(Roche), 1 mM DATP (Roche), 1 unit terminal transferase (Roche) at 37°C. for 5 minutes then denatured at 80° C. for 15 minutes.

3′ cDNA library construction: 2 μg of RNA isolated from either Lotus,soybean, pea or alfalfa tissue was heated to 65° C. for 10 minutes, thensnap cooled on ice. The RNA was reverse transcribed in a 20 μl reactionfor 90 minutes at 42° C. using 10 units of stratascript in 1×RT buffer30 mM dNTPs, 0.1M DTT, 1 U/μl Rnasin and 5 pmoles oligo dt₁₇.

1.3.3 PCR Amplification

Library PCR amplification: Song of 3′ cDNA was PCR amplified in 50 μl of1×PCR buffer (Roche), 20 mM dNTPs 25 pmoles 5′ primer, 25 pmoles 3′primer, 2.5 units Taq DNA polymerase (Roche), 0.25 units pfu turbo DNApolymerase (Stratagene). Cycling conditions were; 30 cycles of 94° C.for 30 seconds, 55° C. for 30 seconds, 72° C. for 2 minutes, using aPerkin Elmer PCR machine. The initial denaturing step (94° C.) wasextended to 2 minutes.

Vector PCR amplification: 1 ng of a vector was PCR amplified in 50 μl of1×PCR buffer (Stratagene), 20 mM dNTPs 25 pmoles 5′ primer, 25 pmoles 3′primer, 5 units pfu turbo DNA polymerase. Cycling conditions were; 30cycles of 94° C. for 30 seconds, 55° C. for 30 seconds, 72° C. for 2minutes. The initial denaturing step (94° C.) was extended to 2 minutes.

5′ Rapid Amplification of cDNA Ends (5′race): 50 ng of 5′ cDNA wascomplemented in 50 μl of 1×PCR buffer, 20 mM dNTPs, 5 pmoles oligo dt₁₇,1.25 units Taq DNA polymerase, 0.125 units pfu turbo DNA polymerase.Conditions were 94° C. for 2 minutes, 42° C. for 2 minutes, 72° C. 45minutes. The cDNA was amplified by adding the following; 25 pmoles 5′R_(O) primer, 25 pmoles primer R_(O), 1.25 units Taq DNA polymerase,0.125 units pfu turbo DNA polymerase to the reverse transcriptionreaction. Cycling conditions were; 30 cycles of 94° C. for 30 seconds,55° C. for 30 seconds, 72° C. 2 minutes. The initial denaturing step wasextended to 2 minutes. 1 μl of this PCR reaction was re-amplified in 50μl of 1×PCR buffer, 20 mM dNTPs 25 pmoles 5′R_(O) primer, 25 pmolesR_(O) primer, 2.5 units Taq DNA polymerase, 0.25 units pfu turbo DNApolymerase. Cycling conditions were; 30 cycles of 94° C. for 30 seconds,55° C. for 30 seconds, 72° C. 30 seconds. The initial denaturing stepwas extended to 2 minutes.

3′ Rapid Amplification of cDNA Ends (3′race): 50 ng of 3′ cDNA wasamplified in 50 μl of 1×PCR buffer, 20 mM dNTPs, 25 pmoles 5′R_(O)primer, 25 pmoles primer R_(O), 2.5 units Taq DNA polymerase, 0.25 unitspfu turbo DNA polymerase. Cycling conditions were; 30 cycles of 94° C.for 30 seconds, 55° C. for 30 seconds, 72° C. 2 minutes. The initialdenaturing step was extended to 2 minutes. 1 μl of this PCR reaction wasre-amplified with 5′R_(I) primer and R_(I) as before with cyclingconditions: 30 cycles of 94° C. for 30 seconds, 55° C. for 30 seconds,72° C. I minute. The initial denaturing step was extended to 2 minutes.

1.3.4 Isolation and Cloning of Amplified Products

Fragments generated by PCR were analysed on an ETBr-1.2% agarose TBE (45mM Tris-borate, 1 mM EDTA) gel. DNA fragments were isolated from the gelusing a Gibco-BRL gel extraction kit according to manufacturer'sinstructions and cloned into a pT7 TA cloning vector (Novagen).

1.3.5 Digesting DNA with Restriction Enzymes

PCR amplifications or 2 μg of plasmid DNA were digested with 10 units ofeach appropriate restriction enzyme (Roche) in the recommended buffer at37° C. for 2 hours. Digests were separated on EtBr-1.2% agarose TBE gel:The desired fragments were excised and purified using the Gibco-BRL gelextraction kit according to manufacturer's instructions.

1.3.6 Construction of Synthetic Linkers

1 μg of sense and anti-sense oligonucleotides were annealed together byheating to 94° C. for 5 minutes in 1× ligation buffer and then cooled toroom temperature over a period of 30 minutes.

1.3.7 De-Phosphorylation of DNA Fragments

Vector DNA fragments were incubated in 50 μl of 1×sip (Roche) bufferwith 0.5 units shrimp intestinal phosphorylase (Roche) at 37° C. for 15minutes and then denatured at 80° C. for 5 minutes.

1.3.8 Sub-Cloning into Vectors

DNA fragments of interest were ligated into appropriate vectors in aratio of 5:1 in a final volume of 20 μl containing 1× ligation buffer(Roche), 2 units of T4 DNA ligase (Roche) at 4-8° C. for 16 hours.

1.3.9 Preparation and Transformation of Competent E. coli

To prepare competent cells a culture of XL1-Blue (from a single colony)was grown up overnight at 37° C., 225 r.p.m. in 10 ml Lennox brothcontaining 12.5 μg/ml tetracycline. 1 ml from this overnight culture wastransferred into 100 mls of fresh, pre-warmed, Lennox broth and culturedfor a further 2 hours until the OD₆₀₀ was in the range 0.3 to 0.6. Thecells were then recovered by centrifugation at 4500 g for 10 minutes at4° C. The cells were washed in 50 ml 100 mM CaCl₂, before resuspendingin a final volume of 5 ml 100 mM CaCl₂. The cells were then placed onice for 1 hour.

Transformations were performed as follows: One-fifth (4 μl) of theligation reaction was added to 200 μl competent cells. The mixture wasincubated on ice for 30 minutes then heat shocked at 42° C. for 40seconds. 300 μl of 2YT was then added to the mixture before incubatingat 37° C., 225 r.p.m. for 30 minutes. The transformations were thenplated out on Lennox agar containing 100 μg/ml carbenicillin or 50 μg/mlkanamyicin and incubated at 37° C. overnight.

1.3.10 Identification and Screening of E. coli Recombinants

Positive transformants were identified by amplifying DNA from a singlecolony in a 50 μl reaction containing the following mixture, 1×pcrbuffer, 0.2 mM dNTPs, 25 pmoles 5′ primer, 25 pmoles 3′ primer, 1.25units Taq DNA polymerase. Cycling conditions were 94° C. for 30 seconds,55° C. for 30 seconds, 72° C. 1 minute, for 30 cycles. The initialdenaturing step was extended to 2 minutes. The pcr amplifications werethen analysed on EtBr-1.2% agarose TBE gels.

1.3.11 Extraction and Purification of Plasmid DNA

Selected colonies were grown up overnight in 50 mls of 2TY brothcontaining either 100 μg/ml carbenicillin or 50 μg/ml kanamycin (asappropriate) at 225 r.p.m. at 37° C. The cells were recovered bycentrifugation at 4500 g for 10 minutes at 4° C. The bacterial pelletwas resuspended in 4 ml of solution 1 (25 mM Tris.Cl pH8.0, 10 mM EDTA),then 8 ml of solution 2 (0.2N NaOH, 1% SDS) was added, and left at roomtemperature for 5 minutes to lyse the cells. 6 ml of ice-cold solution 3(3M potassium, 5M acetate) was added and the mixture incubated on icefor 15 minutes. The bacterial lysate was then centrifuged at 15000 g, 4°C. for 20 minutes, and the supernatant was filtered through 4 layers ofmiracloth (CalBiochem). 10 mls of Isopropanol was added to precipitatethe DNA and the precipitate spun for 15 minutes at 15000 g, at roomtemperature. The pellet was resuspended in 1 ml of TE (10 mM Tris.ClpH7.6, 1 mM EDTA) with 10 μg/ml RNase A and incubated at 50° C. for 30minutes to remove contaminating RNA. The solution was extracted twicewith an equal volume of phenol/chloroform (1:1) then once with an equalvolume of chloroform. The DNA was re-precipitated with 0.1 volume 3MNaAc pH5.2 and 0.7 volume isopropanol, then spun for 5 minutes at 10000g, at room temperature. The pellet was washed in 70% ETOH then air driedand resuspended in TE at a final concentration of 1 μg/μl.

1.4 Vector Construction

Expression vectors were generated containing chalcone reductase (CHR),chalcone isomerase (CHI) and isoflavone synthase (IFS) cDNA's from Pisumsativum, Lotus corniculatus and Glycine max respectively. CHR, CHI andIFS transgenes were placed under the control of the double 35s promoterto give high levels of expression in tobacco tissues.

1.4.1 Construction of Plasmid pPV5LN

To construct pPV5LN, pUC19 was modified as follows. Firstly, plasmidpPV3 was constructed by removing the HindIII/EcoRI multiple cloning sitefrom pUC19 and replacing it with a synthetic DNA fragment, destroyingthe original EcoRI and HindIII sites and introducing SgfI, HindIII,KpnI, EcoRI and XbaI restriction sites. This synthetic fragment wasconstructed by annealing the oligonucleotides 624 and 625 (FIG. 4). Thisresulted in plasmid pPV3.

The KpnI/EcoRI insert from pSJ30 containing the 2×35S-promoter sequenceupstream of an ˜1.9 kb coding sequence, followed by the Nos terminatorsequence was ligated with pPV3 restricted with KpnI/EcoRI. This resultedin plasmid pPV5.

The ˜1.9 kb coding sequence was then removed from pPV5 as a SalI/SacIfragment and replaced by a synthetic DNA fragment introducing NcoI, NheIand MunI restriction sites, while leaving the original SalI/SacI sitesintact. This synthetic fragment was constructed by annealing theoligonucleotides 626 and 627 (FIG. 4). This resulted in plasmid pPV5L.

The sequence immediately 5′ of the start codon ATG in pPV5L (CCACC) wasreplaced by the plant Kozak sequence TAAACC using PCR. Oligonucleotides640 and 641 (FIG. 4) were used to amplify the 189 bp 3′ fragment of the2×35S promoter from vector pCP031 (van Engelen et al., 1994), modifyingthe Kozak sequence via oligonucleotide 641. pCP031 and the amplifiedfragment were then restricted with HindIII/EcoRV and EcoRV/NcoIrespectively before ligation with pPV5L restricted with HindIII-NcoI toreplace the promoter. This resulted in plasmid pPV5LN (FIG. 5).

1.4.2 Construction of Plasmid pPE-2

To construct the plasmid pPE-2, the multiple cloning site of pPV5LN wasmodified by the insertion of three oligonucleotide adapters. First,oligonucleotides 331 and 332 (FIG. 4) were annealed together and ligatedwith plasmid pPV5LN restricted with EcoRI-XbaI. This resulted in plasmidp5LNa. Next, the multiple cloning site from pPV5LN was amplified usingoligonucleotides 248 and 191 (FIG. 4) and the amplification productrestricted with XbaI and EcoRI. This product was then ligated, inconjunction with the annealed product of oligonucleotides 333 and 334(FIG. 4) with p5LNa restricted with NcoI-EcoRI. This resulted in plasmidp5LNb. To construct plasmid pPE-2, oligonucleotides 329 and 330 wereannealed together and ligated with plasmid p5LNb restricted withSfiI-HindIII. This resulted in plasmid pPE-2 (FIG. 5).

1.4.3 Construction of Plasmid pPE-5

To construct the plasmid pPE-5, the multiple cloning site of pSJ34 wasmodified by the insertion of an oligonucleotide adapter. First,oligonucleotides 337 and 338 (FIG. 4) were annealed together and ligatedwith plasmid pSJ34 restricted with HindIII-EcoRI. This resulted inplasmid pPE-5 (FIG. 5).

pSJ34 is a derivative of the binary vector pGPTV-Kan (Becker et al.,1992 Plant Mol. Biol. 20: 1195-1197) in which the BamHI site between thenptII selectable marker and the gene7 poly (A) signal was destroyed by‘filling-in’ with klenow polymerase.

1.4.4 Construction of Plasmid pPE-9 (2×35S+kozak-Lotus CHI-Tnos)

Lotus CHI cDNA was amplified from the lotus 3′ and the 5′ cDNA libraryusing primers 160/323 and 160/321 respectively (FIG. 4); theamplification products were then re-amplified using primers 198/324 and198/322 respectively (FIG. 4). The amplified fragments were separated byelectrophoresis and products 5a.3.19 and 2.11 respectively were clonedinto the vector pT7 and sequenced with primers 152 and 191 (FIG. 4).

To verify the DNA sequence of the amplified fragments primers 386 and387 were used to amplify the complete coding region of the CHI gene (intriplicate) from a lotus 3′ cDNA library. The resultant fragmentsLCHI-A, LCHI-B and LCHI-C were cloned into vector pT7 and sequenced withprimers 152 and 191. Clone LCHI-A was re-amplified with primers 386/403and 402/387, the resultant fragments were digested with NcoI-PstI andPstI-NheI respectively and ligated into NcoI-XbaI opened PE-2 to createthe vector PE-9 (2×35S+Kozak-Lotus CHI-tNOS in PE-2) (FIG. 5).

1.4.5 Construction of Plasmid pPE-11 (2×35S+kozak-Pea CHR-Tnos)

The chalcone reductase cDNA was amplified from a Pisum sativum leaftissue 3′ cDNA library using primers 384 and 385. The resulting 0.98 kbproduct was ligated with the PCR cloning vector pT7Blue [Novagen] andthe sequence verified before further sub-cloning.

The chalcone reductase sequence was then amplified from the pT7Bluevector using oligonucleotides 384/362, 363/398, 399/400 and 401/385(Table 1). The resulting amplification products were restricted withNcoI-NarI, NarI-BamHI, BamHI-MunI, and MunI-NheI respectively beforeligation with pPE-2 restricted with NcoI-XbaI. This resulted in plasmidpPE-11 (FIG. 5).

1.4.6 Construction of Plasmid pPE-15 (2×35S+kozak-Soy IFS-Tnos)

The isoflavone synthase cDNA was amplified from a Glycine max leaftissue cDNA library using primers 339/340, 341/342, and 343/344 (Table1). The resulting amplification products were restricted with NcoI-ApaI,ApaI-SalI, and SalI-NheI respectively before ligation with pPE-2restricted with NcoI-XbaI. This resulted in plasmid pPE-15 (FIG. 5).

1.4.7 Construction of Plant Transformation Vector pPE-120 (CHR-IFS)

The single gene constructs described above were used to construct theplasmid pPE120 as follows. Plasmids pPE-11 and pPE-15 were restrictedwith SalI-EcoRI and HindIII-SalI respectively. The 2×35S+kozak-PeaCHR-Tnos and 2×35S+kozak-Soy IFS-Tnos fragments were then ligated withpPE-5 restricted with HindIII-EcoRI. This resulted in plasmid pPE120(FIG. 5).

1.4.8 Construction of Plant Transformation Vector pPE-125 (CHR-CHI-IFS)

To construct the plant transformation vector pPE125, plasmids pPE-9 andpPE-120 were restricted with SalI. The resulting 2×35S+kozak-LotusCHI-Tnos fragment (from pPE-9) was then ligated with SalI linearisedpPE-120. This resulted in plasmid pPE-125 (FIG. 5).

1.4.10 GPTV Control Plasmid

A GPTV-based binary plasmid, pPE51 (FIG. 5), containing the double CaMV35s promoter and the nos poly(A) signal (Pd35s-Tnos) was used as controlplasmid. This allows direct comparison between transformed controlplants and plants containing the CHR, CHI and IFS constructs generatedvia a tissue culture procedure.

pPE51 was constructed by restricting pPE2 with EcoRI-HindIII. The2×35S+kozak-Tnos fragment was then ligated with pPE5 restricted withHindIII-EcoRI. This resulted in plasmid pPE51 (FIG. 5).

1.5 A. tumefaciens Transformation

Binary plasmids of pPE120, pPE125, pPE130 and pPE51 were introduced intoAgrobacterium tumefaciens strain LBA4404 by high voltage electroporationas described by Shen and Forde (1989). Briefly, electrocompetent cellsof A. tumefaciens were prepared by inoculation of 50 ml of 2×YT medium(Sambrook et al., 1989) and culturing with shaking at 100 rpm at 28° C.until the culture reached an OD₆₀₀ of 0.5-0.7. The cells were cooled onice, harvested by centrifugation and the supernatant discarded. Thecells were then washed successively in 50, 25, 1 and 1 ml of cold 10%(v/v) glycerol before re-suspension in 0.5 ml 10% glycerol.

For transformation, 40 μl of cells were transferred to a pre-cooled 0.2cm electroporation cuvette (Bio-Rad Laboratories). One μl of eitherpPE120 or pCJ102 plasmid DNA was mixed with the cell suspension on iceand an electric pulse applied immediately using a Gene Pulser with Pulsecontroller unit (Bio-Rad). For transformation, the field strength was12.5 kV/cm, a capacitance of 25 μF and resistors of 400-600 ohms inparallel, giving time constants of 8-12 msec. The cells were immediatelytransferred to 1 ml 2×TY and shaken at 29° C. for 2 hours. Aliquots werethen plated onto LB agar supplemented with kanamycin and incubated for2-3 days at 29° C.

The presence and integrity of the plasmids in kanamycin resistant cloneswas established by PCR analysis using pPE120 (GPTV2 and 30035s;340-GPTV1), pPE51 (30035S and GPTV2), pPE125 (GPTV2 and 30035s;340-GPTV1; 248-403; 402-398), pPE130 (GPTV2-30035S) specific primersrespectively (FIG. 4).

1.6 Stable Transformation of Nicotiana tabacum cv SR1

A. tunefaciens cells from PCR positive colonies were used to inoculate a10 ml Lennox media broth containing kanamycin 50 :g/ml and rifampicin 50:g/ml and incubated overnight with shaking (120 rpm) at 29° C. Theovernight culture was centrifuged at 3000 g and the cell pelletresuspended in an equal volume of MS media (3% sucrose). Leaf segmentswere cut from young Nicotiana tabacum L. cv. SR1 leaves from plantsgrown in tissue culture. The leaf segments were placed directly into theA. tumefaciens suspension and co-incubated for 10 minutes.

The leaf segments were then transferred, axial surface down, to feederplates (10 per plate) and placed at 22° C. for 2 days in low light. Theleaf segments were then transferred, axial surface up, to tobaccoshooting media supplemented with hormones, cefotaxime 500 :g/ml andkanamycin 50 :g/ml and placed in a growth room at 24° C. with a 16 hrphotoperiod. After three weeks, callusing segments were transferred tofresh tobacco shooting media in vitro-vent [Melford Laboratories Ltd.]tissue culture vessels. Shoots were then excised from callused leafsegments and placed on tobacco shooting media without hormonescontaining cefotaxime 500 :g/ml and kanamycin 50 :g/ml.

Genomic DNA was isolated from shoots that had rooted and transgenicplants harbouring the constructs were selected following specificamplification of the CHR, CHI, IFS transgenes respectively.

Transgene positive plants were then potted up into a 50% perlite 50%compost mixture and placed in a propagator in a growthroom at 25° C.with a 16 h photoperiod (3000 lux). After 1 week the plants were removedfrom the propagator and subsequently potted up into 5-inch pots.

Petal tissue was harvested from each independent transformant and storedfor subsequent analysis. When flowering had finished, each plant wascut-back and allowed to re-grow to form new flowers, from which seedswere harvested for propagation and analysis.

1.7 Extraction of Flavonoids and Isoflavonoids from Tobacco Tissues

Flavonoids and isoflavonoids were determined as their glycosides or asaglycones by preparing non-hydrolysed and hydrolysed extracts,respectively.

For extraction, tobacco petal tissues were harvested from fully open,mature flowers. To ensure representative analyses, all of the flowers(>10 per plant) were harvested at a similar developmental stage fromeach pPE120, pPE125 and corresponding pPE51 (control) plants. The flowerwas fractionated to remove stamen, carpel and corolla tube tissue andthe remaining petal tissue was then flash frozen in liquid nitrogenbefore being stored at −80° C. The petal tissues (>10) from each plantwere then ground to a fine powder to ensure a homogeneous mix. Analiquot from this mixture was then extracted for 30 min at roomtemperature (˜22° C.) in 80% (v/v) ethanol at 100 mg/700 μl. Followingextraction, the cell debris was removed by filtration through a 0.45 μmMillex-HV filter unit (Millipore Corp, USA). The filtrate was stored at−20° C. prior to HPLC analyses.

For hydrolysed extracts, 40 μl of 12M HCl was added to 360 μl from eachpetal extract, before incubating at 90° C. for 40 min.

Daidzin/genistin standards were hydrolysed under the same conditions asthe petal extracts providing a control for the hydrolysis process.

1.8 Flavonoid and Isoflavonoid Analyses

1.8.1 HPLC Conditions for Flavonoid and Isoflavonoid Analysis

After hydrolysis, an aliquot from each extract was filtered through a0.2 μm PTFE disposable filter (Whatman). The filtrate (20 μl) from wasinjected into the HPLC system (HP1100, Agilent) via an autosamplermaintained at 4° C. The analytical column (Prodigy Phenyl-3, 4.6×150 mm,particle size 5 μm, (Phenomenex) was held at 30° C. Detection was bydiode array, monitoring at 262, 280, and 370 nm. Observed peaks werescanned from 210-550 nm to obtain spectra. Chemstation software (Rev.A.8.03) was used to control the system and collect and analyse data.

Separation of flavonoid and isoflavonoid components within the extractswas performed using a gradient of acetonitrile in 1% acetic acid, at aflow rate of 0.8 ml/min. The gradient of acetonitrile was: 15-37% linearin 22 min, then 37-80% in 2 min, before a hold at 80% for 2 min. Thenthe acetonitrile was reduced from 80-15% in 2 min and held at 15% for 2min prior to next injection.

Absorbance spectra (corrected for baseline spectrum) and retention timeof peaks were compared with those of commercially available flavonoidand isoflavonoid standards.

Calibration curves for quercetin, kaempferol, genistein, daidzein,isoliquiritigenin and liquiritigenin were established to permitquantitation in the hydrolysed tobacco extracts. Levels were calculatedon a fresh weight (μg/g F.wt.) basis. With the HPLC system and softwareused, detection limits in tobacco extracts was about 0.1 μg/ml,corresponding with ˜1.5 μg/g fresh weight. Variation between replicateinjections was less than 5%.

1.8.2 GC-MS Conditions for Flavonoid and Isoflavonoid Analysis

After hydrolysis, 5 ml of 10% Na₂SO₄ was added to an aliquot from eachtissue extract before extraction with 2 ml ethyl acetate. The sample wasthen centrifuged at 1600 g for 1 min. The ethyl acetate layer wasdecanted to a fresh tube and evaporated to dryness under N₂ (<45° C.)

Samples were dissolved in 30 μl pyridine and derivatised by heating with20 μl bis-trifluoroacetamide (BSTFA) at 45° C. for 15 min., 1 μl ofsample was injected onto a CP-Sil 8 CB/MS (25 m×0.25 mm×0.25 μm film) GCcapillary column (Chrompack) through a splitless injector port at 280°C. (Hewlett Packard 5890 gas chromatograph). The oven temperature wasset at a linear temperature gradient from 100-320° C. at 10° C./min witha helium gas flow rate of 1 ml/min. The mass spectrum was monitoredusing a Hewlett Packard 5972A quadruple mass-selective detector set at300° C. (EI) and mass ranges of 175, 184, 383, 398 Daltons for daidzein(selective ion mode); 228, 399, 371, 486 Daltons for genistein(selective ion mode); (219, 307, 371, 457 and 472 daltons forisoliquiritigenin (selective ion mode) and 151, 179, 192, 235, 385, and400 daltons for liquiritigenin (selective ion mode). In addition, massranges of 170-400 Daltons for daidzein, 130-480 Daltons forisoliquiritigenin, 130-410 Daltons for liquiritigenin and 180-490 forgenistein were selected for full scan mode.

1.9 Accumulation of Daidzein in Transgenic N. tabacum EctopicallyExpressing Chalcone Reductase and Isoflavone Synthase:

To determine whether ectopic expression of both chalcone reductase andisoflavone synthase in the non-leguminous plant N. tabacum was able toredirect flavonoid synthesis toward daidzein and/or genistein synthesis,the flavonoid and isoflavonoid profile of petal tissues was determined.This analysis was performed by HPLC using hydrolysed extracts of petaltissue from nineteen pPE120 and six pPE51 transformants.

In the HPLC analysis comparison between hydrolysed petal extracts fromflowers of N. tabacum transformed with either pPE120 or pPE51 indicatedthat in several of the pPE120 transformants a small peak with the sameretention time as the daidzein standard was detected. By contrast, thisHPLC peak was not present in control (pPE51) transformants. To confirmour preliminary identification, this peak was collected from the HPLCand analysed using GC-MS assay. In addition, fractions with thecorresponding retention time were collected from a typical pPE51transformant and from a daidzein standard as controls.

GC-MS analysis showed that the retention time and the relative abundanceof the measured ions (175, 184, 383, and 398 [M⁺]) from the pPE120fraction were similar to those from the authentic daidzein standard(FIG. 6). Furthermore, the fraction from pPE51 showed no GC peak with asimilar retention time or with a similar relative abundance of themeasured ions confirming the absence of daidzein in the controltransformants (FIG. 6).

Quantitation, based on comparison with authentic standards showed thatlevels of daidzein accumulation in pPE120 petal tissues reached up to˜2.75 μg/gFwt (FIG. 7).

1.10 Daidzein Accumulation in Transgenic N. tabacum Expressing ChalconeReductase, Isoflavone Synthase and Chalcone Isomerase.

To determine whether concomitant expression of chalcone reductase andisoflavone synthase in conjunction with a legume chalcone isomerase inthe non-leguminous plant N. tabacum was able to enhance the level ofdaidzein accumulation, the flavonoid and isoflavonoid profile of petaltissues was determined. This analysis was performed by HPLC usinghydrolysed extracts of petal tissue from twelve pPE125 and six pPE51transformants.

In the HPLC assay comparison between hydrolysed petal extracts fromflowers of N. tabacum transformed with either pPE125 or pPE51 indicatedthat for several of the pPE125 transformants a peak with the sameretention time as the daidzein standard was detected. By contrast, thispeak was not present in control (pPE51) transformants. To confirm ourpreliminary identification, the peak corresponding to daidzein wascollected from the HPLC and analysed using GC-MS assay. In addition,fractions with the corresponding retention time were collected from atypical pPE51 transformant and from a daidzein standard as controls.

GC-MS analysis showed that the retention time and the relative abundanceof the measured ions (175, 184, 383, and 398 [M⁺]) from the pPE125fraction were similar to those from the authentic daidzein standard.Furthermore, the fraction from pPE51 showed no peak with a similarretention time or with a similar relative abundance of the measured ionsconfirming the absence of daidzein. Quantitation, based on comparisonwith authentic standards showed that levels of daidzein accumulation inpPE125 petal tissues reached up to 246.7 μg/gFwt (˜4934 μg/gDwt) (FIG.8).

EXAMPLE 2 Transformation of Lettuce Stable Transformation of LactucaSativa L. cv Lollo Rossa, Bijou, Muscara & Revolution

A. tumefaciens cells from PCR positive colonies were used to inoculate a10 ml Lennox media broth containing kanamycin 50 μg/ml and rifampicin 50μg/ml and incubated overnight with shaking (120 rpm) at 29° C. Theovernight culture was centrifuged at 3000 g and the cell pelletresuspended in an equal volume of UM media and a 1:10 (v/v) dilutionused for transformation.

Cotyledons were cut from 7-day old Lactuca Sativa L. seedlings grown intissue culture. The abaxial surface of the cotyledons was scored with ascalpel-blade before placing directly into the A. tumefaciens suspensionand co-incubated for 10 minutes.

The cotyledons were then transferred, abaxial surface down, tosolidified UM media supplemented with 3% (w/v) sucrose overlayed withone filter paper (8 per plate) and placed at 25° C. for 2 days. Thecotyledons were then transferred, axial surface up, to solidified MSmedia supplemented with 3% (w/v) sucrose, 0.04 mgl⁻¹ NAA, 0.5 mgl⁻¹ BAP,100 μg/ml cefotaxime, 500 μg/ml carbenicillin and 50 μg/ml kanamycin andplaced in a growth room at 25° C. with a 16 hr photoperiod. The explantswere transferred to fresh medium every 14 days. After eight weeks,regenerating explants were transferred to solidified MS mediasupplemented with 0.11% (w/v) MES, 100 μg/ml cefotaxime and 50 μg/mlkanamycin.

Genomic DNA was isolated from shoots that had rooted and transgenicplants harbouring the constructs were selected following specificamplification of the CHR, CHI & IFS transgenes respectively.

Transgene positive plants were then transferred to 9 cm diameter potscontaining Levington M3 compost mixed with John Innes No. 3 & perlite(3:3:2) and placed in a propagator in a growthroom at 25° C. with a 16hr photoperiod. After 1 week the plants were removed from the propagatorand maintained at 25° C. with a 16 hr photoperiod.

Leaf tissue harvested from each independent transformant and is storedat −80° C. for subsequent flavonoid and isoflavonoid analyses aspreviously described.

EXAMPLE 3 Transformation of Potato

Stable Transformation of Solanum tuberosum L. cv. Desiree

A. tumefaciens cells from transgene positive (PCR) colonies were used toinoculate a 20 ml Lennox media broth containing kanamycin 50 μg/ml andrifampicin 50 μg/ml and incubated for 3-days with shaking (120 rpm) at29° C. Following incubation, this culture was centrifuged at 3000 g andthe cell pellet resuspended in 25 ml MS media (pH5.8) supplemented with3% (w/v) sucrose.

Leaves were cut from 4-week old Solanum tuberosum L. plants, grown intissue culture, and placed axial surface up onto solidified L3 medium[MS basal salts supplemented with 1.6% glucose, 0.8% agar, pH5.8]supplemented with 0.02 mg/l NAA, 20 mg/l GA₃, 2 mg/l Zeatin riboside]and placed at 23° C. for 2 days.

The excised leaves were then placed directly into the A. tumefacienssuspension and co-incubated for 10 minutes. Following co-incubation, theleaves were ‘blotted-dry’ and transferred, axial surface up, to feederplates (solidified L3 media overlayed with 2 ml of tobacco cellssuspension over which one filter paper was placed) and placed indarkness at 23° C. for 2 days. The leaf explants were then transferred,axial surface up, to solidified L3 media supplemented 0.02 mg/l NAA, 20mg/l GA3, and 500 μg/ml cefotaxime and placed in a growth room at 23/Cwith a 16 hr photoperiod for four days. The leaf explants were thentransferred to fresh L3 medium supplemented with 0.02 mg/l NAA, 20 mg/lGA3, and 500 μg/ml cefotaxime and 100 mg/l kanamycin every 14 days.After approximately eight weeks, shoots (˜1.5 cm) were excised from theregenerating explants and transferred to solidified MS mediasupplemented with 1% (w/v) sucrose, 0.8% agar, 500 μg/ml cefotaxime and100 μg/ml kanamycin.

Genomic DNA was isolated from shoots that had rooted and transgenicplants harbouring the constructs were selected following specificamplification of the CHR, CHI & IFS transgenes respectively.

Minitubers were initiated from each transgene positive plant by transferof ˜3 cm long leaf node to MS media supplemented with 8% (w/v) sucroseand 0.8% agar and maintaining in darkness at 25° C. Minitubers wereharvested from each independent transformant and stored at −80° C. forsubsequent flavonoid and isoflavonoid analyses.

EXAMPLE 4 Food Product: Skin Appearance Benefits from IsoflavoneConsumption

The investigation was designed as a double blind placebo controlledstudy with 33 female post-menopausal volunteers. The participants wererandomised in a parallel design into two groups to receive foods withand without functional ingredients for a period of 12 weeks in total.For the duration of the study the subjects had to avoid soya containingfoods and stop taking vitamins, minerals or other dietary supplements.

The study comprised two phases. Firstly, a “run-in” or “washout” phasewhen subjects consumed placebo foods for two weeks. Secondly, anintervention phase when subjects were randomly allocated to consumefoods (2 low-calorie food bars per day) containing functionalingredients or placebo foods for a further 10 weeks.

Study foods were provided as a low-calorie bar, The bars were small(serving size 29 g) and provided on average 108 calories and 3.1 g fat.

Each functional bar contained:

Soya isoflavones 20 mg Green tea polyphenols 100 mg Gamma-linolenic acid240 mg Carotenoids 0.25 mg Vitamin A 300 μg Vitamin C 60 mg Vitamin E7.5 mg Vitamin B2 0.55 mg Vitamin B3 7 mg Vitamin B6 0.75 mg Vitamin D 5μg Folate 200 μg Zinc 7.5 mg Calcium 600 mg PABA 120 mg

The placebo foods contained the PABA (para-aminobenzoic acid) but noneof the functional ingredients. PABA was added as a compliance marker toall the bars.

Consumption of the bars containing micronutrients of which theisoflavones are considered to most efficacious, resulted in a range ofskin health and appearance benefits: i. Improved skin appearance andreduced signs of ageing due to reduced wrinkle height; ii. Improvedfirmness and skin tone; iii. Softer and smoother skin; iv. A lesssensitive skin, that makes one feel better about their skin; v. Improvedoverall antioxidant status of the body and skin.

The statistical significance of each of the skin benefits or serumchanges after ten weeks intervention is listed below:

Week 10 ‘p’ Parameter value i. Wrinkle height (replicas) <0.078 ii.Firmness (indent value) <0.075 iii. Softness/Smoothness (Coefficient ofrestitution) <0.15 iv. Sensitive skin (questionnaire) <0.05 v. Serumantioxidant status (TEAC) <0.065

1. A genetically modified plant or part thereof comprising daidzein and/or derivatives thereof, wherein said plant or part thereof does not naturally produce isoflavones and is active in both flavonol and anthocyanin biosynthesis and comprises: (a) a first nucleotide sequence encoding a chalcone reductase comprising an amino acid with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2; and (b) a second nucleotide sequence encoding an isoflavone synthase comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO:
 4. 2. A genetically modified plant or part thereof according to claim 1, further comprising a third nucleotide sequence encoding a chalcone isomerase comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO:
 6. 3. A genetically modified plant or part thereof according to claim 1 wherein (i) the first nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 1, and (ii) the second nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:
 3. 4. A genetically modified plant or part thereof according to claim 2 wherein the third nucleotide comprises the nucleotide sequence of SEQ ID NO:
 5. 5. A genetically modified plant or part thereof according claim 4 wherein the third nucleotide sequence consists of the nucleotide sequence as shown in SEQ ID NO:
 5. 6. A genetically modified plant or part thereof according to claim 1 wherein said plant or part thereof is selected from the group consisting of tobacco, Lactuca sp., broccoli, asparagus, red cabbage, potato, spinach, rhubarb, red onion, shallot, aubergine, radish, Swiss chard, purple basil, watermelon and berries.
 7. A food product comprising a genetically modified plant or part thereof according to claim
 1. 8. A food product according to claim 7 wherein said food product is selected from the group consisting of packaged mixed salad, soup, spread, sauce, fruit bar and ice cream.
 9. A method for the production of a food product or nutritional supplement comprising culturing the genetically modified plant or part thereof according to claim 1 under conditions suitable for expression of a chalcone reductase or isoflavone synthase.
 10. A method for the production of a food product or nutritional supplement comprising culturing the genetically modified plant or part thereof according to claim 2 under conditions suitable for expression of a chalcone reductase or isoflavone synthase.
 11. A process for increasing the content of daidzein and/or derivatives thereof in a plant or part thereof, wherein said process comprises: (i) selecting a non-isoflavone producing plant wherein said plant or part thereof is active in both anthocyanin and flavonol biosynthesis; and (ii) genetically modifying said plant to incorporate one or more nucleotide sequences encoding a chalcone reductase comprising an amino acid with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2 with chalcone reductase activity and one or more nucleotide sequences encoding a isoflavone synthase comprising the an amino acid with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4 with isoflavone synthase activity so as to increase the activity of chalcone reductase and isoflavone synthase in said plant or part thereof.
 12. A process according to claim 11, wherein said process further comprises genetically modifying said plant or part thereof to incorporate one or more nucleotide sequences encoding a chalcone isomerase comprising an amino acid with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 6 capable of catalyzing the conversion of 4,2′,4′-trihydroxchalcone to 7,4′-dihydroxyflavanone so as to increase the activity of the chalcone isomerase.
 13. A process according to claim 11, wherein (i) one or more nucleotide sequences encoding a chalcone reductase comprises a nucleotide sequence as shown in SEQ ID NO: 1, and (ii) one or more nucleotide sequences encoding a isoflavone synthase comprises a nucleotide sequence as shown in SEQ ID NO:
 3. 14. A process according to claim 12, wherein said one or more nucleotide sequences encoding a chalcone isomerase comprises a nucleotide sequence as shown in SEQ ID NO:
 5. 15. A process according to claim 14, wherein said one or more nucleotide sequences encoding a chalcone isomerase consists of a nucleotide sequence as shown in SEQ ID NO:
 5. 16. A process according to claim 11 wherein said plant is selected from the group consisting of tobacco, Lactuca sp., broccoli, asparagus, red cabbage, potato, spinach, rhubarb, red onion, shallot, aubergine, radish, Swiss chard, purple basil, watermelon and berries.
 17. A genetically modified plant or part thereof according to claim 1, wherein the first nucleotide sequence encodes a chalcone reductase comprising the amino acid sequence of SEQ ID NO:
 2. 18. A genetically modified plant or part thereof according to claim 1, wherein the first nucleotide sequence encodes a chalcone reductase consisting of the amino acid sequence of SEQ ID NO:
 2. 19. A genetically modified plant or part thereof according to claim 1, wherein the second nucleotide sequence encodes a isoflavone synthase comprising the amino acid sequence of SEQ ID NO:
 4. 20. A genetically modified plant or part thereof according to claim 1, wherein the second nucleotide sequence encodes a isoflavone synthase consisting of the amino acid sequence of SEQ ID NO:
 4. 21. A genetically modified plant or part thereof according to claim 2, wherein the third nucleotide sequence encodes a chalcone isomerase comprising the amino acid sequence of SEQ ID NO:
 6. 22. A genetically modified plant or part thereof according to claim 3, wherein the first nucleotide sequence consists of the nucleotide sequence of SEQ ID NO:
 1. 23. A genetically modified plant or part thereof according to claim 3, wherein the second nucleotide sequence consists of the nucleotide sequence of SEQ ID NO:
 3. 24. The process of claim 11, wherein the chalcone reductase comprises the amino acid sequence of SEQ ID NO:
 2. 25. The process of claim 11, wherein the chalcone reductase consists of the amino acid sequence of SEQ ID NO:
 2. 26. The process of claim 11, wherein the isoflavone synthase comprises the amino acid sequence of SEQ ID NO:
 4. 27. The process of claim 11, wherein the isoflavone synthase consists of the amino acid sequence of SEQ ID NO:
 4. 28. The process of claim 12, wherein the chalcone isomerase comprises the amino acid sequence of SEQ ID NO:
 6. 29. The process of claim 12, wherein the nucleotide sequence encoding the chalcone isomerase consists of the amino acid sequence of SEQ ID NO:
 6. 30. The process of claim 13, wherein the nucleotide sequence encoding the chalcone reductase consists of the nucleotide sequence of SEQ ID NO:
 1. 31. The process of claim 13, wherein the nucleotide sequence encoding the isoflavone synthase consists of the nucleotide sequence of SEQ ID NO:
 3. 